Insights into the lignocellulosic physiology of the yeast pathogen Cryptococcus neoformans var. grubii

Botes, Angela (2014-04)

Thesis (PhD)--Stellenbosch University, 2014.

Thesis

ENGLISH ABSTRACT: Cryptococcus neoformans (Sanfelice) Vuillemin is an opportunistic pathogen that causes cryptococcal meningitis, predominantly in immuno-compromised individuals, particularly in those suffering from human immuno virus (HIV) or acquired immuno-deficiency syndrome (AIDS). This basidiomycetous yeast species is sub-divided into two main varieties, C. neoformans var. neoformans (serotype D) and C. neoformans var. grubii (serotype A), as well as a rare hybrid variety, C. neoformans (serotype AD). The global incidence of cryptococcosis among AIDS sufferers is approximately 30 % with 90 % of these cases being attributed to serotype A. Cryptococcus neoformans has been isolated from numerous environmental sources including guano, soil, and particularly decaying wood and tree hollows. Infection occurs when particles originating from these environments are inhaled. The ecological niche of C. neoformans was thought to be avian guano; however, recent findings indicate that the true ecological niche may rather be woody material. Representatives of this species, particularly C. neoformans var. grubii, were found to grow on agar plates containing carboxymethyl cellulose as carbon source; however, little is known about its ability to degrade hemi-cellulose. As such, the overall aim of this project was to study the interactions of C. neoformans var. grubii ATCC H99 with cellulosic and hemi-cellulosic materials. Growth studies revealed that C. neoformans var. grubii ATCC H99 was capable of utilizing carboxymethyl cellulose, glucomannan and galactomannan as sole carbon sources. This yeast also assimilated simple degradation products of lignocellulose such as L-arabinose, D-galactose, D-glucose, D-mannose, L-rhamnose and D-xylose. D-Mannose and D-glucose resulted in the highest maximum specific growth rates. Screening the genome of C. neoformans var. grubii ATCC H99 resulted in the identification of three putative cellulases, specifically an endo-glucanase (EC7) and two cellulases (CC1 and CC6). Evaluation of the deduced amino acid sequences indicated that all three enzymes belong to glycoside hydrolase family 5 (GHF5). Phylogenetic analyses revealed that the three enzymes grouped in distinct clades with other GHF5 members. Automated homology modeling of the three-dimensional structure revealed that CC1 and CC6 displayed the classical (α/β)8 TIM barrel fold associated with GHF5. Modeling of EC7 did not produce the classic GHF5 structure, suggesting that this enzyme may be classed in a separate GHF. Green fluorescent protein (GFP) tagging confirmed that CC6 forms part of the cryptococcal secretome. Real-time quantitative PCR (qPCR) analyses indicated that the three proteins responded differently on a transcriptional level in the presence of various carbohydrates. Cellulase CC6 displayed the most dynamic expression profile, indicating up-regulation in the presence of mannose, galactose and cellobiose. The use of Acacia mearnsii debris and the aqueous extract thereof also resulted in a significant up-regulation of all three enzymes. This confirms previous findings that the woody phyloplane is a natural habitat of C. neoformans var. grubii. In the presence of mucin, the transcription of CC6 was up-regulated. Similar to laccase and urease, CC6 may aid the survival of C. neoformans within the human respiratory system. Understanding the carbohydrate metabolic regulatory system and its impact on virulence would increase our overall knowledge of this pathogen‘s survival capabilities and infection strategies.

AFRIKAANSE OPSOMMING: Cryptococcus neoformans (Sanfelice) Vuillemin is 'n opportunistiese patogeen wat kriptokokkale meningitis veroorsaak, hoofsaaklik in immuun-gekompromiteerde individue en veral diegene wat die menslike immunovirus (MIV) of verworwe immuniteitsgebreksindroom (VIGS) onderlede het. Hierdie basidiomisete gisspesie word in twee hoof variëteite verdeel, naamlik C. neoformans var. neoformans (serotipe D) en C. neoformans var. grubii (serotipe A), asook 'n skaars hibried-variëteit, C. neoformans (serotipe AD). Die wêreldwye voorkoms van kriptokokkose onder VIGS-lyers is ongeveer 30 %, met 90 % van hierdie gevalle wat aan serotipe A toegeskryf kan word. Cryptococcus neoformans is uit talle omgewingsbronne geïsoleer, insluitende guano, grond en veral verrottende hout en boomholtes. Infeksie vind plaas wanneer partikels uit hierdie omgewings ingeasem word. Voëlguano is voorheen as die ekologiese nis van C. neoformans beskou, maar onlangse bevindinge dui aan dat die ware ekologiese nis eerder houtagtige materiaal mag wees. Verteenwoordigers van hierdie spesie, veral C. neoformans var. grubii, kon op agar plate te groei wat karboksimetielsellulose as koolstofbron bevat; min is egter bekend oor sy vermoë om hemisellulose af te breek. Die hoofdoel van hierdie projek was dus om die interaksies van C. neoformans var. grubii ATCC H99 met sellulosiese en hemi-sellulosiese materiale te bestudeer. Groeistudies het getoon dat C. neoformans var. grubii ATCC H99 in staat was om karboksimetielsellulose, glukomannaan en galaktomannaan as enigste koolstofbronne te benut. Hierdie gis het ook eenvoudige afbraakprodukte van lignosellulose, L-arabinose, D-galaktose, D-glukose, D-mannose, L-rhamnose en D-xylose geassimileer. D-Mannose en D-glukose het die hoogste maksimum spesifieke groeitempo‘s tot gevolg gehad. Die sifting van C. neoformans var. grubii ATCC H99 se genoom het gelei tot die identifisering van drie moontlike sellulases: 'n endo-glukanase (EC7) en twee sellulases (CC1 en CC6). Evaluering van afgeleide die aminosuur-volgordes het aangedui dat al drie ensieme aan die glikosied hidrolase familie 5 (GHF5) behoort. Filogenetiese analises het getoon dat die drie ensieme in duidelike taksonomiese groepe saam met ander lede van die GHF5 voorkom. Outomatiese homologie-modellering van die drie-dimensionele struktuur het getoon dat CC1 en CC6 die klassieke (α/β)8 TIM vaatjie-vou, wat met GHF5 geassosieer word, vertoon. Modellering van EC7 het nie die klassieke GHF5 struktuur geproduseer nie, wat daarop mag dui dat hierdie ensiem moontlik in 'n afsonderlike GHF geklassifiseer kan word. Groen fluoresserende protein (GFP) merking het bevestig dat CC6 deel van die kriptokokkale sekretoom vorm. Intydse kwantitatiewe PCR (qPCR) analises het getoon dat die drie proteïene verskillend op 'n transkripsievlak in die teenwoordigheid van verskillende koolhidrate reageer. Sellulase CC6 het die mees dinamiese uitdrukkingsprofiel getoon, wat dui op op-regulering in die teenwoordigheid van mannose, galaktose en sellobiose. Die gebruik van Acacia mearnsii molm en die waterige ekstrak daarvan het ook 'n beduidende op-regulering van hierdie drie ensieme tot gevolg gehad. Dit bevestig vorige bevindings dat die houtagtige plantoppervlak 'n natuurlike habitat vir C. neoformans var. grubii is. Die transkripsie van CC6 is ook in die teenwoordigheid van musien op-gereguleer. Net soos lakkase en urease, mag CC6 tot die oorlewing C. neoformans in die menslike asemhalingstelsel bydra. 'n Begrip van die koolhidraat metaboliese reguleringsisteem en die impak daarvan op virulensie sal ons algehele kennis van hierdie patogeen se vermoë en strategie om te oorleef, verbeter.

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